Chapter 10 Use of Intein‐Mediated Protein Ligation Strategies for the Fabrication of Functional Protein Arrays

Chattopadhaya, Souvik; Bakar, Farhana B. Abu; Yao, Shao Q.

doi:10.1016/s0076-6879(09)62010-3

Cited by 8 publications

(6 citation statements)

References 53 publications

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“…By comparison, the MOrPH-forming ability of 3 stems from the superior intein splicing properties of its 2-amino-benzylthiol, a structure which has never been described in the context of thioester-or intein-mediated ligations. 15 Clearly, such a structure preserves the nucleophilicity of the benzylic thiol while placing the amino group at a viable distance for acyl transfer via a six-membered ring intermediate.…”

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confidence: 99%

Diverse organo-peptide macrocyclesvia a fast and catalyst-free oxime/intein-mediated dual ligation

et al. 2012

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Diverse organo-peptide macrocyclesvia a fast and catalyst-free oxime/intein-mediated dual ligation

et al. 2012

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“…Splicing afforded the fluorescent scFv which was purified to homogeneity exploiting the biotin hand [ 89 ], while fluorescent probe allowed the in vivo antibody detection. A “mirror” approach, useful to label protein N-terminus, has also been described and applied to the imaging in living cells [ 3 ]. In this work, the intein-mediated protein splicing allowed the in viv o generation of the target protein bearing an N-terminal Cys residue.…”

Section: Fluorescent Labelingmentioning

confidence: 99%

“…Spectroscopic techniques rely tightly on protein labeling strategies by which the chemical structure of a protein is modified through the introduction of biophysical probes, such as fluorophores or isotopes. A wide collection of protein labeling approaches have been developed in recent years [ 1 , 2 , 3 , 4 ], leading to great discoveries and innovations. In particular, the introduction of native chemical ligation (NCL) methodologies for chemical synthesis of proteins marked a breakthrough in protein and peptide chemistry, with a strong impact on chemical biology and biophysical applications [ 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

Semi-Synthesis of Labeled Proteins for Spectroscopic Applications

et al. 2013

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Since the introduction of SPPS by Merrifield in the 60s, peptide chemists have considered the possibility of preparing large proteins. The introduction of native chemical ligation in the 90s and then of expressed protein ligation have opened the way to the preparation of synthetic proteins without size limitations. This review focuses on semi-synthetic strategies useful to prepare proteins decorated with spectroscopic probes, like fluorescent labels and stable isotopes, and their biophysical applications. We show that expressed protein ligation, combining the advantages of organic chemistry with the easy and size limitless recombinant protein expression, is an excellent strategy for the chemical synthesis of labeled proteins, enabling a single protein to be functionalized at one or even more distinct positions with different probes.

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“…Although a substitution level of one biotin per ligand is recommended, the chemical methods described often result in multilabeled compounds, thus impairing the validity and significance of the SPR results [26]. Intein-mediated protein splicing combined with native chemical ligation using a cysteine biotin derivative is a more specific approach that overcomes this set of problems [39]. Moreover, the Escherichia coli ( E. coli ) biotin ligase (BirA) can be used to biotinylate site specifically a ligand fused to the recognition sequence of BirA [40].…”

Section: Setting Up the Experimentsmentioning

confidence: 99%

Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance

Ritzefeld

Sewald

2012

Journal of Amino Acids

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Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

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Chapter 10 Use of Intein‐Mediated Protein Ligation Strategies for the Fabrication of Functional Protein Arrays

Cited by 8 publications

References 53 publications

Diverse organo-peptide macrocyclesvia a fast and catalyst-free oxime/intein-mediated dual ligation

Diverse organo-peptide macrocyclesvia a fast and catalyst-free oxime/intein-mediated dual ligation

Semi-Synthesis of Labeled Proteins for Spectroscopic Applications

Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance

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